---
title: "Inferring Immune Interactions in Breast Cancer"
author: "Orian Stapleton"
date: "2024-01-08"
output:
    BiocStyle::html_document:
        toc: yes
        toc_depth: 3
        number_sections: yes
vignette: >
    %\VignetteIndexEntry{Inferring Immune Interactions in Breast Cancer}
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding{UTF-8}
---

```{r global.options, include = FALSE}
knitr::opts_knit$set(
    collapse = TRUE,
    comment = "#>",
    fig.align   = 'center'
)

knitr::opts_chunk$set(out.extra = 'style="display:block; margin:auto;"')

```

# Overview

SpaceMarkers leverages latent feature analysis of the spatial components of 
transcriptomic data to identify biologically relevant molecular interactions 
between cell groups.This tutorial will use the latent features from CoGAPS to 
look at pattern interactions in a Visium 10x breast ductal carcinoma spatial 
transcriptomics dataset.

# Installation

```{r eval = FALSE}
if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("SpaceMarkers")
```
```{r message = FALSE, warning = FALSE}
library(SpaceMarkers)
```

# Setup
## Links to Data

The data that will be used to demonstrate SpaceMarkers' capabilities is a human
breast cancer spatial transcriptomics dataset that comes from Visium.The CoGAPS
patterns as seen in the manuscript 
[Atul Deshpande et al.](https://doi.org/10.1016/j.cels.2023.03.004) 
will also be used

```{r}
main_10xlink <- "https://cf.10xgenomics.com/samples/spatial-exp/1.3.0"
counts_folder <- "Visium_Human_Breast_Cancer"
counts_file <- "Visium_Human_Breast_Cancer_filtered_feature_bc_matrix.h5"
counts_url<-paste(c(main_10xlink,counts_folder,counts_file), collapse = "/")
sp_folder <- "Visium_Human_Breast_Cancer"
sp_file <- "Visium_Human_Breast_Cancer_spatial.tar.gz"
sp_url<-paste(c(main_10xlink,sp_folder,sp_file),collapse = "/")
```

The functions require that some files and directories with the same name be 
unique. Therefore any downloads from a previous runs will be removed.
```{r}
unlink(basename(sp_url))
files <- list.files(".")[grepl(counts_file,list.files("."))]
unlink(files)
unlink("spatial", recursive = TRUE)
```


## Extracting Counts Matrix
### load10xExpr

Here the counts matrix will be obtained from the h5 object on the Visium site 
and genes with less than 3 counts are removed from the dataset.This can be
achieved with the load10XExpr function.

```{r}
download.file(counts_url,basename(counts_url), mode = "wb")
counts_matrix <- load10XExpr(visiumDir = ".", h5filename = counts_file)
good_gene_threshold <- 3
goodGenes <- rownames(counts_matrix)[
    apply(counts_matrix,1,function(x) sum(x>0)>=good_gene_threshold)]
counts_matrix <- counts_matrix[goodGenes,]
files <- list.files(".")[grepl(basename(counts_url),list.files("."))]
unlink(files)
```

## Obtaining CoGAPS Patterns

In this example the latent features from CoGAPS will be used to identify 
overlapping genes with SpaceMarkers. Here the featureLoadings (cells) and 
samplePatterns (genes) for both the expression matrix and CoGAPS matrix need to 
match.

```{r}
cogaps_result <- readRDS(system.file("extdata","CoGAPS_result.rds",
    package="SpaceMarkers",mustWork = TRUE))
features <- intersect(rownames(counts_matrix),rownames(
    slot(cogaps_result,"featureLoadings")))
barcodes <- intersect(colnames(counts_matrix),rownames(
    slot(cogaps_result,"sampleFactors")))
counts_matrix <- counts_matrix[features,barcodes]
cogaps_matrix<-slot(cogaps_result,"featureLoadings")[features,]%*%
    t(slot(cogaps_result,"sampleFactors")[barcodes,])
```

## Obtaining Spatial Coordinates
### load10XCoords

The spatial coordinates will also be pulled from Visium for this dataset. 
These are combined with the latent features to demonstrate how cells for each 
pattern interact in 2D space. The data can be extracted with the 
**load10XCoords()** function

```{r}
download.file(sp_url, basename(sp_url), mode = "wb")
untar(basename(sp_url))
spCoords <- load10XCoords(visiumDir = ".", version = "1.0")
rownames(spCoords) <- spCoords$barcode
spCoords <- spCoords[barcodes,]
spPatterns <- cbind(spCoords,slot(cogaps_result,"sampleFactors")[barcodes,])
head(spPatterns)
```
For demonstration purposes we will look at two patterns; Pattern_1 (immune cell)
and Pattern_5 (invasive carcinoma lesion). Furthermore we will only look at the
relationship between a pre-curated list of genes for efficiency.
```{r}
data("curated_genes")
spPatterns<-spPatterns[c("barcode","y","x","Pattern_1","Pattern_5")]
counts_matrix <- counts_matrix[curated_genes,]
cogaps_matrix <- cogaps_matrix[curated_genes, ]
```

# Executing SpaceMarkers

## SpaceMarker Modes

SpaceMarkers can operate in 'residual'or 'DE' (DifferentialExpression) mode. 
In an ideal world the overlapping patterns identified by SpaceMarkers would be a
homogeneous population of cells and the relationship between them would be 
linear. However, due to confounding effects of variations in cell density and 
common cell types in any given region, this is not always true.

To account for these confounding effects, the 'residual' mode compares the 
feature interactions between the expression matrix and the reconstructed latent
space matrix. The features with the highest residual error are reported. 
The genes are then classified according to regions of overlapping vs exclusive 
influence. The default mode is 'residual' mode.

Suppose the feature (gene) information is not readily available and only the 
sample (cells) latent feature patterns with P-values are available? 
This is the advantage of 'DE' mode. Where residual mode assesses the non-linear 
effects that may arise from confounding variables, 'DE' mode assesses simple 
linear interactions between patterns directly from expression. DE mode also 
compares genes from regions of overlapping vs exclusive influence but does not 
consider residuals from the expression matrix as there is no matrix 
reconstruction with the latent feature matrix.

### Residual Mode

SpaceMarkers identifies regions of influence using a gaussian kernel outlier 
based model. The reference pattern (Pattern_1 in this case) is used as the prior
for this model. SpaceMarkers then identifies where the regions of influence are 
interacting from each of the other patterns as well as where they are mutually 
exclusive.

getSpatialParameters: This function identifies the optimal width of the gaussian
distribution (sigmaOpt) as well as the outlier threshold around the set of spots
(thresOpt) for each pattern.These parameters minimize the spatial 
autocorrelation residuals of the spots in the regions of influence. 

getSpatialParameters took approximately 5 minutes on a MacBook Pro Quad-Intel 
Core i5 processor with 16 GB of memory. Therefore we will load this data from
a previous run. The spPatterns object is the sole parameter for this function if
you would like to run this yourself

```{r}
data("optParams")
optParams
```

getInteractingGenes: This function identifies the regions of influence and 
interaction as well as the genes associated with these regions. A non-parametric
Kruskal-Wallis test is used to identify statistically significant genes in any 
one region of influence without discerning which region is more significant. 
A post hoc Dunn's Test is used for analysis of genes between regions and can 
distinguish which of two regions is more significant. If 'residual' mode is 
selected the user must provide a reconstructed matrix from the latent feature
matrix. The matrix is passed to the 'reconstruction' argument and can be left 
as NULL for 'DE' mode. The 'data' parameter is the original expression matrix. 
The 'spPatterns' argument takes a dataframe with the spatial coordinates of 
each cell as well as the patterns. The spatial coordinate columns must contain 
the labels 'x' and 'y' to be recognized by the function.

```{r}
SpaceMarkers <- getInteractingGenes(data = counts_matrix,
                                    reconstruction = cogaps_matrix,
                                    optParams = optParams,
                                    spPatterns = spPatterns,
                                    refPattern = "Pattern_1",
                                    mode ="residual",analysis="overlap")

```

NB: When running getInteractingGenes some warnings may be generated. 
The warnings are due to the nature of the 'sparse' data being used. 
Comparing two cells from the two patterns with identical information is 
redundant as SpaceMarkers is identifying statistically different expression for
interactions exclusive to either of the two patterns and a region that is due 
to interaction between the given two patterns. Also, if there are too many 
zeros in the genes (rows) of those regions, the columns are dropped as there 
is nothing to compare in the Kruskal Wallis test.


```{r}
print(head(SpaceMarkers$interacting_genes[[1]]))
print(head(SpaceMarkers$hotspots))
```

The output is a list of data frames with information about the interacting genes
of the refPattern and each pattern from the CoGAPS matrix 
(interacting_genes object). 
There is also a data frame with all of the regions of influence for any two of 
patterns (the hotspotRegions object).

For the 'interacting_genes' data frames, the first column is the list of genes 
and the second column says whether the statistical test were done vsPattern_1,
vsPattern_2 or vsBoth. The remaining columns are statistics
for the Kruskal-Wallis test and the post hoc Dunn's test.The SpaceMarkersMetric
column is a product of sums of the Dunn's statistics and 
is used to rank the genes.

### DE Mode

As described previously 'DE' mode only requires the counts matrix and spatial 
patterns and not the reconstructed CoGAPS matrix. It identifies simpler 
molecular interactions between regions.

```{r}
SpaceMarkers_DE <- getInteractingGenes(
    data=counts_matrix,reconstruction=NULL,
    optParams = optParams,
    spPatterns = spPatterns,
    refPattern = "Pattern_1",
    mode="DE",analysis="overlap")
```

### Differences between Residual Mode and DE Mode

One of the first things to notice is the difference in the number 
of genes identified between the two modes. Here we will use the genes 

```{r}
residual_p1_p5<-SpaceMarkers$interacting_genes[[1]]
DE_p1_p5<-SpaceMarkers_DE$interacting_genes[[1]]
```

```{r}
paste(
    "Residual mode identified",dim(residual_p1_p5)[1],
        "interacting genes,while DE mode identified",dim(DE_p1_p5)[1],
        "interacting genes",collapse = NULL)
```
DE mode produces more genes than residual mode because the matrix of 
residuals highlights less significant differences for confounding genes 
across the spots.The next analysis will show where the top genes rank in each 
mode's list if they are identified at all. A function was created that will take
the top 20 genes of a reference list of genes and compare it to the entire list
of a second list of genes. The return object is a data frame of the gene, the
name of each list and the ranking of each gene as compared to the reference 
list. If there is no gene identified in the second list compared to the 
reference it is classified as NA.

```{r}

compare_genes <- function(ref_list, list2,ref_name = "mode1",
                            list2_name = "mode2", sub_slice = NULL){
    ref_rank <- seq(1,length(ref_list),1)
    list2_ref_rank <- which(list2 %in% ref_list)
    list2_ref_genes <- list2[which(list2 %in% ref_list)]
    ref_genes_only <- ref_list[ !ref_list  %in% list2_ref_genes ]
    mode1 <- data.frame("Gene" = ref_list,"Rank" = ref_rank,"mode"= ref_name)
    mode2 <- data.frame("Gene" = c(list2_ref_genes, ref_genes_only),"Rank" = c(
        list2_ref_rank,rep(NA,length(ref_genes_only))),"mode"= list2_name)
    mode1_mode2 <- merge(mode1, mode2, by = "Gene", all = TRUE) 
    mode1_mode2 <- mode1_mode2[order(mode1_mode2$Rank.x),]
    mode1_mode2 <- subset(mode1_mode2,select = c("Gene","Rank.x","Rank.y"))
    colnames(mode1_mode2) <- c("Gene",paste0(ref_name,"_Rank"),
                                paste0(list2_name,"_Rank"))
    return(mode1_mode2)
}
```

```{r}
res_to_DE <- compare_genes(head(residual_p1_p5$Gene, n = 20),DE_p1_p5$Gene,
                            ref_name="residual",list2_name="DE")
DE_to_res <- compare_genes(head(DE_p1_p5$Gene, n = 20),residual_p1_p5$Gene,
                            ref_name = "DE",list2_name = "residual")
```

### Comparing residual mode to DE mode

```{r}
res_to_DE
```

Here we identify the top 20 genes in 'residual' mode and their corresponding 
ranking in DE mode.HLA-DRB1 is the only gene identified in 
residual mode and not in DE mode. The other genes are ranked relatively high
in both residual and DE mode.

### Comparing DE mode to residual mode

```{r}
DE_to_res
```

Recall that DE mode looks at the information encoded in the latent feature space
and does not filter out genes based on any confounders between the counts matrix
and latent feature matrix as is done in 'residual' mode. Therefore there are
more genes in DE mode not identified at all in residual mode. 

There is some agreement with interacting genes between the two methods but there
are also quite a few differences. Therefore, the selected mode can significantly
impact the downstream results and should be taken into consideration based on 
the specific biological question being answered and the data available.

## Types of Analyses

Another feature of the SpaceMarkers package is the type of analysis that can be
carried out, whether 'overlap' or 'enrichment' mode. The major difference 
between the two is that enrichment mode includes genes even if they did not 
pass the post-hoc Dunn's test. These additional genes were included to enable a
more statistically powerful pathway enrichment analysis and understand to a 
better extent the impact of genes involved each pathway. 
Changing analysis = 'enrichment' in the getInteractingGenes function will enable
this.

```{r}
SpaceMarkers_enrich <- getInteractingGenes(data = counts_matrix,
                                    reconstruction = cogaps_matrix,
                                    optParams = optParams,
                                    spPatterns = spPatterns,
                                    refPattern = "Pattern_1",
                                    mode ="residual",analysis="enrichment")
SpaceMarkers_DE_enrich <- getInteractingGenes(
    data=counts_matrix,reconstruction=NULL,
    optParams = optParams,
    spPatterns = spPatterns,
    refPattern = "Pattern_1",
    mode="DE",analysis="enrichment")
residual_p1_p5_enrichment<-SpaceMarkers_enrich$interacting_genes[[1]]$Gene
DE_p1_p5_enrichment<-SpaceMarkers_DE_enrich$interacting_genes[[1]]$Gene

```


### Residual Mode and DE Mode - Enrichment

The data frames for the Pattern_1 x Pattern_5 will be used to compare the 
results of the enrichment analyses

```{r}
enrich_res_to_de<-compare_genes(
    head(DE_p1_p5_enrichment, 20),
    residual_p1_p5_enrichment,
    ref_name="DE_Enrich",list2_name = "res_Enrich")
enrich_res_to_de
```
The ranks differ alot more here because now genes that were not previously 
ranked are assigned a score.

```{r}
overlap_enrich_de<-compare_genes(
    head(DE_p1_p5_enrichment,20),
    DE_p1_p5$Gene,
    ref_name="DE_Enrich",
    list2_name="DE_Overlap")
overlap_enrich_de
```
The enrichment and overlap analysis are in great agreement for DE mode.
Typically, you may see slight changes among genes lower in the ranking. This is 
especially important where genes that do not pass the Dunn's test for 
interactions between any of the other two patterns in the overlap analysis are 
now ranked in enrichment analysis. The Pattern_1 x Pattern_5 column for these 
genes is labelled as FALSE. 

Here is an example of the statistics for such genes. 
```{r}
tail(SpaceMarkers_DE_enrich$interacting_genes[[1]])
```
A similar trend can be observed when comparing the overlap and enrichment
analysis in residual mode.
```{r}
overlap_enrich_res<-compare_genes(
    head(residual_p1_p5$Gene, 20),
    residual_p1_p5_enrichment,
    ref_name ="res_overlap",list2_name="res_enrich")
overlap_enrich_res
```

# Visualizing SpaceMarkers

The differences between the gene interactions of Pattern_1 and 
Pattern_5 can be visualized in various ways to view 
both the magnitude and location of expression in space. In this analysis the top
2-3 genes in residual mode from Pattern_5 only vs Pattern_1, Pattern_1 only vs 
Pattern_5 and the interacting region vs both Pattern_1 and Pattern_5 will be
compared.

## Loading Packages

The following libraries are required to make the plots:

```{r message = FALSE, warning=FALSE}
library(Matrix)
library(rjson)
library(cowplot)
library(RColorBrewer)
library(grid)
library(readbitmap)
library(dplyr)
library(data.table)
library(viridis)
library(hrbrthemes)
library(ggplot2)
```

## Code Setup

This first function below can visualize the locations of these patterns on a 
spatial grid. The code has been adopted from 10xgenomics:
(support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/rkit)

```{r}
geom_spatial <-  function(mapping = NULL,
    data = NULL,
    stat = "identity",
    position = "identity",
    na.rm = FALSE,
    show.legend = NA,
    inherit.aes = FALSE,...) {
    GeomCustom <- ggproto(
        "GeomCustom",
        Geom,
        setup_data = function(self, data, params) {
            data <- ggproto_parent(Geom, self)$setup_data(data, params)
            data
        },
        
        draw_group = function(data, panel_scales, coord) {
            vp <- grid::viewport(x=data$x, y=data$y)
            g <- grid::editGrob(data$grob[[1]], vp=vp)
            ggplot2:::ggname("geom_spatial", g)
        },
        
        required_aes = c("grob","x","y")
        
    )
    
    layer(geom = GeomCustom,mapping = mapping,data = data,stat = stat,position=
                position,show.legend = show.legend,inherit.aes =
                inherit.aes,params = list(na.rm = na.rm, ...)
    )
}
```

Some spatial information for the breast cancer dataset is required.

```{r}
sample_names <- c("BreastCancer")
image_paths <- c("spatial/tissue_lowres_image.png")
scalefactor_paths <- c("spatial/scalefactors_json.json")
tissue_paths <- c("spatial/tissue_positions_list.csv")

images_cl <- list()

for (i in 1:length(sample_names)) {
    images_cl[[i]] <- read.bitmap(image_paths[i])
}

height <- list()

for (i in 1:length(sample_names)) {
    height[[i]] <-  data.frame(height = nrow(images_cl[[i]]))
}

height <- bind_rows(height)

width <- list()

for (i in 1:length(sample_names)) {
    width[[i]] <- data.frame(width = ncol(images_cl[[i]]))
}

width <- bind_rows(width)

grobs <- list()
for (i in 1:length(sample_names)) {
    grobs[[i]]<-rasterGrob(images_cl[[i]],width=unit(1,"npc"),
                            height=unit(1,"npc"))
}

images_tibble <- tibble(sample=factor(sample_names), grob=grobs)
images_tibble$height <- height$height
images_tibble$width <- width$width


scales <- list()

for (i in 1:length(sample_names)) {
    scales[[i]] <- rjson::fromJSON(file = scalefactor_paths[i])
}

```

It is also helpful to adjust spot position by scale factor and format some of 
the tissue information.

```{r}
bcs <- list()
for (i in 1:length(sample_names)) {
    bcs[[i]] <- read.csv(tissue_paths[i],col.names=c(
        "barcode","tissue","row","col","imagerow","imagecol"), header = FALSE)
    bcs[[i]]$imagerow <- bcs[[i]]$imagerow * scales[[i]]$tissue_lowres_scalef
    # scale tissue coordinates for lowres image
    bcs[[i]]$imagecol <- bcs[[i]]$imagecol * scales[[i]]$tissue_lowres_scalef
    bcs[[i]]$tissue <- as.factor(bcs[[i]]$tissue)
    bcs[[i]]$height <- height$height[i]
    bcs[[i]]$width <- width$width[i]
}
names(bcs) <- sample_names
```

Adding umi per spot, total genes per spot and merging the data

```{r}
matrix <- list()
for (i in 1:length(sample_names)) {
    matrix[[i]] <- as.data.frame(t(as.matrix(counts_matrix)))
}
umi_sum <- list()
for (i in 1:length(sample_names)) {
    umi_sum[[i]] <- data.frame(barcode =  row.names(matrix[[i]]),
                                sum_umi = Matrix::rowSums(matrix[[i]]))
}
names(umi_sum) <- sample_names

umi_sum <- bind_rows(umi_sum, .id = "sample")
gene_sum <- list()

for (i in 1:length(sample_names)) {
    gene_sum[[i]] <- data.frame(barcode=row.names(
        matrix[[i]]),sum_gene=Matrix::rowSums(matrix[[i]] != 0))

}
names(gene_sum) <- sample_names
gene_sum <- bind_rows(gene_sum, .id = "sample")
bcs_merge <- bind_rows(bcs, .id = "sample")
bcs_merge <- merge(bcs_merge,umi_sum, by = c("barcode", "sample"))
bcs_merge <- merge(bcs_merge,gene_sum, by = c("barcode", "sample"))

```

Specifying a continuous scale and colors before plotting

```{r}
myPalette <- function(numLevels) {
    return(colorRampPalette(c("blue","yellow"))(numLevels))}
```

Extracting top 3 genes ...

```{r}
gene_list <- c()
sp_genes <- SpaceMarkers$interacting_genes[[1]]
interactions <- unique(sp_genes$`Pattern_1 x Pattern_5`)
n_genes <- 3
for (g in 1:length(interactions)){
    
    df <- sp_genes %>% dplyr::filter(
        sp_genes$`Pattern_1 x Pattern_5` == interactions[g] & abs(
        sp_genes$Dunn.zP2_P1) > 1 )
    df <- df[!is.na(df$Gene),]
    valid_genes <- min(nrow(df),n_genes)
    print(paste0("Top ",valid_genes," genes for ",interactions[g]))
    print(df$Gene[1:valid_genes])
    gene_list <- c(gene_list,df$Gene[1:valid_genes])
    
}

```

Visualize expression spatially

```{r message = FALSE, warning=FALSE}

plots <- list()
# default size = 1.75, stroke = 0.5
for (g in gene_list){
    for (i in 1:length(sample_names)) {
        plots[[length(plots)+1]] <- bcs_merge %>%dplyr::filter(
            sample ==sample_names[i]) %>% bind_cols(as.data.table(
            matrix[i])[,g, with=FALSE]) %>% ggplot(aes_string(
                x='imagecol', y='imagerow', fill=g, alpha = g)) +geom_spatial(
                data=images_tibble[i,], aes(grob=grob), x=0.5, y=0.5)+
            geom_point(shape = 21, colour = "black", size = 1.1, stroke = 0.2)+
            coord_cartesian(expand=FALSE)+scale_fill_gradientn(
                colours = myPalette(100))+xlim(0,max(bcs_merge %>%dplyr::filter(
                    sample ==sample_names[i]) %>% select(width)))+ylim(max(
                        bcs_merge %>%dplyr::filter(sample ==sample_names[i])%>%
                            select(height)),0)+xlab("") +ylab("") + ggtitle(
                                sample_names[i])+
            theme_set(theme_bw(base_size = 10))+
            theme(
                panel.grid.major=element_blank(),
                panel.grid.minor = element_blank(),
                panel.background = element_blank(),
                axis.line = element_line(colour="black"),
                axis.text=element_blank(),axis.ticks = element_blank())
    }
}
```

This next block of code can visualize the gene expression in each region using 
box plots.

```{r}
region <- SpaceMarkers$hotspots[,1]
region <- ifelse(!is.na(region)&!is.na(SpaceMarkers$hotspots[,2]),
                    "Interacting",ifelse(!is.na(region),region,
                                            SpaceMarkers$hotspots[,2]))
region <- factor(region, levels = c("Pattern_1","Interacting","Pattern_5"))
plist <- list()
mplot2 <- t(as.matrix(counts_matrix[,!is.na(region)]))
mplot2 <- as.data.frame(as.matrix(mplot2))
mplot2 <- cbind(mplot2,region = region[!is.na(region)])
for (ii in 1:length(gene_list)){
    plist[[ii]]<- mplot2 %>% ggplot( aes_string(x='region',y=gene_list[ii],
                                                fill='region'))+geom_boxplot()+
        scale_fill_viridis(discrete = TRUE,alpha=0.6)+
        geom_jitter(color="black",size=0.4,alpha=0.9)+theme_ipsum()+
        theme(legend.position="none",plot.title = element_text(size=11),
                axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1))+
        ggtitle(paste0(gene_list[ii]," Expression (Log)")) + xlab("") 
}
```

## SpaceMarkers Visualiztions

### Interacting region (vsBoth)

This category compares the interacting region to both Pattern_1 and Pattern_5 
exclusively

Below there are box plots and spatial heatmaps to help visualize the expression
of individual genes across different patterns. The two main statistics used to 
help interpret the expression of genes across the patterns are the KW 
statistics/pvalue and the Dunn's test. In this context the null hypothesis of 
the KW test is that the expression of a given gene across all of the spots is 
equal. The post hoc Dunn's test identifies how statistically significant the 
difference in expression of the given gene is between two patterns. The Dunn's 
test considers the differences between specific patterns and the KW test 
considers differences across all of the spots without considering the specific
patterns.

#### Table of statistics

```{r}
head(residual_p1_p5 %>% dplyr::filter(
    sp_genes$`Pattern_1 x Pattern_5` == "vsBoth"),n_genes)
```

#### Visualizations

```{r, message=FALSE, warning=FALSE, dpi=36, fig.width=12, fig.height=5}
plot_grid(plotlist = list(plist[[1]],plots[[1]]))
plot_grid(plotlist = list(plist[[2]],plots[[2]]))
```

On the spatial heatmap, Pattern_1 takes up most of the top half of the spatial
heatmap, followed by the interacting region along the diagonal and finally 
Pattern_5 in the bottom left corner. APOC1 is expressed across all patterns but
is especially strong in the interacting pattern. IGHE is highly specific to the
interacting pattern. The Dunn.pval_1_Int is lower for IGHE compared to APOC1 
indicating more significance in the interacting region vs the other two regions.
However, the overall SpaceMarkersMetric for APOC1 is higher and so it is 
ranked higher than IGHE.


```{r, message=FALSE, dpi=36, warning=FALSE, fig.width=12, fig.height=5}
plot_grid(plotlist = list(plist[[3]],plots[[3]]))
```

The spatial heatmap for TGM2 shows fairly high expression interacting region 
relative to Pattern_1 or Pattern_5 by itself similar but less so than IGHE. 
as well with high expression in the interacting pattern vs the other 
two patterns.

### Pattern_5 Only vs Pattern_1 (including Pattern_1 in interacting region)


#### Table of statistics

```{r}
head(sp_genes %>% dplyr::filter(
    sp_genes$`Pattern_1 x Pattern_5` == "vsPattern_1"),n_genes - 1 )
```

#### Visualizations

```{r, message=FALSE, dpi=36, warning=FALSE, fig.width=12, fig.height=5}
plot_grid(plotlist = list(plist[[4]],plots[[4]]))
plot_grid(plotlist = list(plist[[5]],plots[[5]]))
```

Unlike the previous three genes the KW pvalues and Dunn's pvalues are relatively
high so the distinction will not appear as pronounced on the expression map.

### Pattern_1 Only vs Pattern_5 (including Pattern_5 in interacting region)

#### Table of statistics

```{r}
head(sp_genes %>% dplyr::filter(
    sp_genes$`Pattern_1 x Pattern_5`=="vsPattern_5"),n_genes - 1)

```

#### Visualizations

```{r, message=FALSE, dpi=36, warning=FALSE, fig.width=12, fig.height=5}
plot_grid(plotlist = list(plist[[6]],plots[[6]]))
plot_grid(plotlist = list(plist[[7]],plots[[7]]))
```

The expression of these genes across interaction is harder to distinguish on
either plot. Although, they pass the initial KW-test, the post-hoc Dunn's test
p-values are high. This lack of distinction highlights the fact that 
SpaceMarkers performs best for identifying interactions for two latent 
individual latent Patterns interacting with each other. In other words, the 
vsBoth genes are ranked highest in the list, while the vsPattern_1 and 
vsPattern_5 genes, which look at an individual Pattern interaction with another
already interacting region, are ranked lower in the SpaceMarkers list.

# Removing Directories

```{r}
unlink(basename(sp_url))
unlink("spatial", recursive = TRUE)
files <- list.files(".")[grepl(basename(counts_url),list.files("."))]
unlink(files)
```

# References

Deshpande, Atul, et al. "Uncovering the spatial landscape of molecular 
interactions within the tumor microenvironment through latent spaces." 
Cell Systems 14.4 (2023): 285-301.

“Space Ranger.” Secondary Analysis in R -Software -Spatial Gene Expression -
Official 10x Genomics Support, 
support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/rkit. 
Accessed 22 Dec. 2023. 

## load10XExpr() Arguments

```{r echo=FALSE}
parameters = c('visiumDir', 'h5filename')
paramDescript = c('A string path to the h5 file with expression information',
                    'A string of the name of the h5 file in the directory')
paramTable = data.frame(parameters, paramDescript)
knitr::kable(paramTable, col.names = c("Argument","Description"))

```

## load10XCoords() Arguments

```{r echo=FALSE}
parameters = c('visiumDir', 'resolution')
paramDescript = c(
'A path to the location of the the spatial coordinates folder.',
'String values to look for in the .json object;lowres or highres.')
paramTable = data.frame(parameters, paramDescript)
knitr::kable(paramTable, col.names = c("Argument","Description"))

```

## getSpatialParameters() Arguments

```{r echo=FALSE}
parameters = c('spPatterns')
paramDescript = c('A data frame of spatial coordinates and patterns.')
paramTable = data.frame(parameters, paramDescript)
knitr::kable(paramTable, col.names = c("Argument","Description"))

```

## getInteractingGenes() Arguments

```{r echo=FALSE}
parameters = c(
        'data','reconstruction', 'optParams','spPatterns',
            'refPattern','mode', 'minOverlap','hotspotRegions','analysis')
paramDescript = c(
        'An expression matrix of genes and columns being the samples.',
        'Latent feature matrix. NULL if \'DE\' mode is specified',
        'A matrix of sigmaOpts (width) and the thresOpt (outlierthreshold)',
        'A data frame that contains of spatial coordinates and patterns.',
        'A string of the reference pattern for comparison to other patterns',
        'A string specifying either \'residual\' or \'DE\' mode.',
        'A value that specifies the minimum pattern overlap. 50 is the default',
        'A vector of patterns to compare to the \'refPattern\'',
        'A string specifying the type of analysis')
paramTable = data.frame(parameters, paramDescript)
knitr::kable(paramTable, col.names = c("Argument","Description"))

```

```{r}
sessionInfo()
```
